Identification and Characterization of a Second Chromophore of DNA Photolyase from Thermus thermophilus HB27

Takumi Ueda,Akira Kato,Seiki Kuramitsu,Hiroaki Terasawa,Ichio Shimada

doi:10.1074/jbc.m507972200

Abstract

Cyclobutane pyrimidine dimer (CPD) photolyases use light to repair CPDs. For efficient light absorption, CPD photolyases use a second chromophore. We purified Thermus thermophilus CPD photolyase with its second chromophore. UV-visible absorption spectra, reverse-phase HPLC, and NMR analyses of the chromophores revealed that the second chromophore of the enzyme is flavin mononucleotide (FMN). To clarify the role of FMN in the CPD repair reaction, the enzyme without FMN (Enz-FMN(-) and that with a stoichiometric amount of FMN (Enz-FMN(+)) were both successfully obtained. The CPD repair activity of Enz-FMN(+) was higher than that of Enz-FMN(-), and the CPD repair activity ratio of Enz-FMN(+) and Enz-FMN(-) was dependent on the wavelength of light. These results suggest that FMN increases the light absorption efficiency of the enzyme. NMR analyses of Enz-FMN(+) and Enz-FMN(-) revealed that the binding mode of FMN is similar to that of 7,8-didemethyl-8-hydroxy-5-deazariboflavin in Anacystis nidulans CPD photolyase, and thus a direct electron transfer between FMN and CPD is not likely to occur. Based on these results, we concluded that FMN acts as a highly efficient light harvester that gathers light and transfers the energy to FAD.

Highlights

Acid (MTHF) and 7,8-didemethyl-8-hydroxy-5-deazariboflavin (8-HDF) as second chromophores, respectively [5, 8]
The crystal structure of T. thermophilus cyclobutane pyrimidine dimer (CPD) photolyase lacks a second chromophore, it contains a pocket that is similar to the 8-HDF-binding site of A. nidulans CPD photolyase [14]
Development of a Purification Protocol Designed to Retain the Second Chromophore—T. thermophilus CPD photolyase was reportedly purified by heat treatment and Blue Sepharose chromatography [19, 24]

Summary

Introduction

Acid (MTHF) and 7,8-didemethyl-8-hydroxy-5-deazariboflavin (8-HDF) as second chromophores, respectively [5, 8]. The crystal structures of the CPD photolyases from E. coli, A. nidulans, and Thermus thermophilus have been solved (Protein Data Bank codes 1DNP, 1QNF, and 1IQR, respectively) [12,13,14,15,16] These structures share a similar overall fold consisting of an ␣/␤ domain and a helical domain. The crystal structures of the CPD photolyases from E. coli and A. nidulans contain MTHF and 8-HDF, respectively, in the ␣/␤ domain [12, 13, 15, 16], and the second chromophores are ϳ15 Å away from the FAD. The crystal structure of T. thermophilus CPD photolyase lacks a second chromophore, it contains a pocket that is similar to the 8-HDF-binding site of A. nidulans CPD photolyase [14]. Based on our characterization of Enz-FMN(ϩ) and Enz-FMN(Ϫ), we conclude that FMN functions as a highly efficient light harvester that gathers light and transfers the energy to FAD

Methods

Results

Conclusion